The potential advantages of peptide arrays, i.e., arrays composed of a plurality of peptidic molecules, are appreciated by researchers, scientists and clinicians. Peptide arrays enable high-throughput screening of compounds that may interact with one or more peptides in the array in a particular manner. For example, an array of peptidic molecules potentially suitable as ligands for a particular biological receptor may be prepared and “screened” with respect to that receptor. Also, arrays of antibodies may be used to screen for multiple pathogenic antigens in a patient sample. Combinations of proteins may also be used to screen for molecules, which interact or are part of a similar metabolic pathway. In addition, peptide arrays can be employed by clinicians to determine whether or not a patient has developed antibodies to particular peptidic antigens. The promise of peptidic arrays, however, has been not been fully realized. This is in large part due to manufacturing challenges, but other problems have been encountered as well.
One proposed method for manufacturing peptide arrays involves the use of piezoelectric technology, in particular, inkjet printing technology. For example, U.S. Pat. No. 6,015,880 to Baldeschwieler et al. is directed to array preparation and in situ oligomer synthesis using a multistep process, wherein the oligomers synthesized may be oligopeptides. A microdrop of a first reagent in liquid form is applied through a single jet of a multiple jet reagent dispenser to a locus on a surface chemically prepared to react with and covalently attach the reagent. The multiple jet reagent dispenser is then displaced relative to the surface, or the surface is displaced with respect to the dispenser, and at least one microdrop containing either the first reagent or a second reagent from a different dispenser jet is applied to a second locus on the substrate, which, again, is chemically prepared to react with and covalently bind the second reagent. Optionally, the second step is repeated using either the first or second reagents, or different reagents in liquid form from different dispenser jets, wherein the process results in an oligomer, such as an oligopeptide, covalently attached to the substrate surface.
Ordinary inkjet technology, however, suffers from a number of drawbacks. Often, inkjet technology involves heating or using a piezoelectric element to force a fluid through a nozzle in order to direct the ejected fluid onto a surface. Thus, the fluid may be exposed to a surface exceeding 200° C. before being ejected, and most, if not all, peptidic molecules, including proteins, degrade under such extreme temperatures. In addition, forcing peptidic molecules through nozzles creates shear forces that can alter molecular structure. Nozzles are subject to clogging, especially when used to eject a macromolecule-containing fluid, and the use of elevated temperatures exacerbates the problem because liquid evaporation results in deposition of precipitated solids on the nozzles. Clogged nozzles, in turn, can result in misdirected fluid or ejection of improperly sized droplets. Finally, ordinary inkjet technology employing a nozzle for fluid ejection generally cannot be used to deposit arrays with feature densities comparable to those obtainable using photolithography or other techniques commonly used in semiconductor processing.
Photolithographic techniques have also been proposed for use in the manufacture peptidic arrays. For example, U.S. Pat. Nos. 5,143,854 and 5,405,783 to Pirrung et al., and U.S. Pat. Nos. 5,445,934 and 5,744,305 to Fodor et al. describe solid phase synthesis of oligomers, including peptidic oligomers, employing photolithographic techniques. These techniques, however, require a large number of masking steps, resulting in a relatively low overall yield, and are prohibitively expensive. In addition, photolithographic techniques often leave behind small amounts of photolithographic processing materials, e.g., photolabile groups. Furthermore, the purity of the peptidic molecules within the array is relatively low, given that truncated proteins result from missed steps (e.g., failure of a photolabile group to be removed), and imprecise masking placement results in misplaced material.
Both approaches inkjet and photolithographic processes generally rely on in situ preparation of the peptidic molecules. These techniques are generally unsuitable for producing high density arrays of even moderately sized peptidic molecules. For example, in situ synthesis of peptides using photolithographic techniques could require over 100 masks for a peptide only six amino acids in length. Furthermore, there is no guarantee that proteins synthesized in situ will adopt the secondary and tertiary structure necessary for biological activity.
A number of patents have described the use of acoustic energy in printing. For example, U.S. Pat. No. 4,308,547 to Lovelady et al. describes a liquid drop emitter that utilizes acoustic principles in ejecting droplets from a body of liquid onto a moving document to form characters or bar codes thereon. A nozzleless inkjet printing apparatus is used wherein controlled drops of ink are propelled by an acoustical force produced by a curved transducer at or below the surface of the ink. In contrast to inkjet printing devices, nozzleless fluid ejection devices described in the aforementioned patent are not subject to clogging and the disadvantages associated therewith, e.g., misdirected fluid or improperly sized droplets.
The applicability of nozzleless fluid ejection has generally been appreciated for ink printing applications. Development of ink printing applications is primarily economically driven by printing cost and speed for acceptable text. For acoustic printing, development efforts have therefore focused on reducing printing costs rather than improving quality, and on increasing printing speed rather than accuracy. For example, U.S. Pat. No. 5,087,931 to Rawson is directed to a system for transporting ink under constant flow to an acoustic ink printer having a plurality of ejectors aligned along an axis, each ejector associated with a free surface of liquid ink. When a plurality of ejectors is used instead of a single ejector, printing speed generally increases, but controlling fluid ejection, specifically droplet placement, becomes more difficult.
U.S. Pat. No. 4,797,693 to Quate describes an acoustic ink printer for printing polychromatic images on a recording medium. The printer is described as comprising a combination of a carrier containing a plurality of differently colored liquid inks, a single acoustic printhead acoustically coupled to the carrier for launching converging acoustic waves into the carrier, an ink transport means to position the carrier to sequentially align the differently colored inks with the printhead, and a controller to modulate the radiation pressure used to eject ink droplets. This printer is described as designed for the realization of cost savings. Because two droplets of primary color, e.g., cyan and yellow, deposited in sufficient proximity will appear as a composite or secondary color, the level of accuracy required is fairly low and inadequate for biomolecular array formation. Such a printer is particularly unsuitable for in situ synthesis requiring precise droplet deposition and consistent placement, so that the proper chemical reactions occur. That is, the drop placement accuracy needed to effect perception of a composite secondary color is much lower than is required for chemical synthesis at photolithographic density levels. Consequently, an acoustic printing device that is suitable for printing visually apprehensible material is inadequate for microarray preparation. Also, this device can eject only a limited quantity of ink from the carrier before the liquid meniscus moves out of acoustic focus and drop ejection ceases. This is a significant limitation with biological fluids, which are typically far more costly and rare than ink. The Quate et al. patent does not address how to use most of the fluid in a closed reservoir without adding additional liquid from an external source.
Thus, there is a need in the art for improved acoustic fluid ejection methods having sufficient droplet ejection accuracy so as to enable the preparation of high density peptidic arrays without the disadvantages associated with photolithographic techniques or inkjet printing devices relying on a nozzle or relatively large volumes of materials for droplet ejection. Specifically, acoustic fluid ejection devices as described herein provide improved control over the spatial direction of fluid ejection without the disadvantages of lack of flexibility and uniformity associated with photolithographic techniques or inkjet printing devices.